Wednesday, April 25, 2012

Viva Voce #2

As I said in my previous post Viva Voce #1, my university practical exams are going on, out of the 4 practicals, now I completed 3 :) Today was a day of E.coli, as I got E.coli with all experiments :)
Microbio is my favorite favorite subject, and the lab, I love to be in microbio lab with my microbial buddies :) :P Here we go with my microbiology practical exam :)

I prepared well for my lab exam yesterday night. I made myself clear with all the concepts behind lab techniques and experiments. Had good confidence :) as this is my favorite lab :)
I was given with a question paper saying:

Major experiment: 
Inoculate 10% of E.coli culture in nutrient broth of ph5 and check the growth rate by measuring turbidity and plot a graph.

Minor experiment: 
Perform gram staining of the given culture, observe shape, color and interpret the results.

:) :) :)))) I got a biggggggggg smile when I saw this easy question paper and started with my experiment :)
I cleaned and sterilized the laminar air hood, took my pH5 nutrient broth, cuvettes, pipette. For accounting the turbidity spectrophotometer is used generally by us. So, I took blank as the broth with out any inoculation in a cuvette and I inoculated the broth with given E.coli culture.

After inoculation, I took that inoculated broth in a cuvette for taking OD at 0 minute. I kept the broth flask in shaker at 37 degree celsius. Oh, gosh... :( I forgot to take two blanks :( I realised this only when I started towards the spec :(  What to do.. I was blinking for a minute, then I checked the cupboard for sterile nutrient broth of pH5, to my goodness I found one more flask of sterile pH5 nutrient broth. (Again I got a biggg smile...)

I took another blank from that sterile broth :) and took O.D. I took O.D of the inoculated broth for every 30 minutes, say at 30,60,90,120,150 minutes.

E.coli normally grows at optimum pH of 7. So, in my pH 5 broth, I expected that the growth would decrease with time. But, to my surprise it increased. Growth was increasing with time. I was confused :(
Then, side by side, in the gap of 30 minutes in taking O.D, I completed gram staining too. I was given with E.coli for Gram staining too. Got, beautiful pink rods :) interpreted that E.coli is a gram negative, hence it stains pink :)  ( What a great experiment :P :P )

In between the external examiner called, "Register number 05..."
oo.. I'm that 05.. I rushed to him with my answer sheets. He got my sheets and started probing it.. He started shooting questions then, Here we go with my Viva voce #2

External examiner: Your getting increased growth with time, do you think, optimum pH of E.coli is 5?
Me: No sir, optimum pH of E.coli is 7.

External examiner: Then, your getting increased growth? Justify this? 
Me: (Thought for a while) Sir, it might be due to contamination with some other species which grows at pH5.
( He was convinced with this answer)
E.coli - Gram staining

External examiner: List some preservation techniques?
Me: Stab culture, Glycerol stock, Lyophilisation.

External examiner: What's the purpose of Safranin in Gram staining?
Me: It is a counter stain. Gram positive retains crystal violet while decorising with ethanol, while Gram negative loses it. So, to give a contrast , Safranin is used. Gram positives won't take up safranin.

External examiner: So.. Crystal violet is a basic dye or acidic?
Me: It's a basic dye, sir.

External examiner: Why a basic dye for staining bacteria? why not an acidic?
Me: Sir, normally bacteria have negative charge, so, using an acidic dye which is also negative charged, there will be repulsion and there won't be staining.

External examiner: okay, that's it. you may go.

He was satisfied with my answers :)  An easy Viva voce once again :)

Then I continued with my experiment, got increase of growth, drawn graph and gave inference as " The increase in growth might be due to contamination of the culture with other species which grows at pH5"

And we were given with some 4 questions:
Here are my 4 questions:

1) Interpret the spotter 1. ( Mam gave an EMB - Eosin methylene blue agar plate) 
My interpretation:
The given plate is an EMB - Eosin methylene blue agar plate - divided into two halves.
In one half, there was growth showing metallic zinc color - indicating that it was E.coli culture.
In another half no growth was obtained, indicating that half was inoculated with a gram positive bacteria.
Principle: EMB agar is a selective media, it allows only gram negatives to grow and E.coli gives metallic zinc with EMB agar.

2) How will you prepare 1ml of 1:5 dilution culture? 
Ans: 0.2 ml of culture in 0.8 ml of broth

3) You are isolating 5 different S.aureus cultures, whether it is necessary to check susceptibility for all the five? Why?
Ans: It is necessary to check susceptibility for all the 5, because one may have resistance and other may not have (  when Isolated from different sources)

4) A simple calculation for calculating phenol coefficient was given. I did it right. I don't remember the numbers given, so, I share just the formula: 

Phenol coefficient = (Greatest dilution of disinfectant that kills microbe in 10 mins but not 5 mins) /
(Greatest dilution of phenol that kills microbe in 10 mins but not 5 mins)

Finished successfully and submitted my paper, came out with satisfaction :) :) :)
Just one more practical exam tomorrow, just it is Communication skills practicals! :) :) Yaaaiii.. yaaii.. did well in all the practicals :) Have a good time :) :)) 

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